Mutations in the Foxp3 gene are associated with lymphoproliferation, atopy, autoimmunity and early lethality in human and mice. Foxp3 is an essential gene for the development and function of regulatory T cells. Selection of regulatory T cells in the thymus is a critical event in the establishment of a healthy immune system. Numerous studies highlighted the importance of high affinity interactions between T cell receptors (TCR) and their ligands in the selection of Foxp3+ regulatory T cells. We generated TCR transgenic (Tg) mice from Foxp3+ T cells and found out that the TCR has an instructive role in determining Foxp3 expression;however, selection of Foxp3+ individual clones in the thymus is limited by a very small niche. We will study the selection of Foxp3+ cells in the thymus as indicated in the following three aims. In Aim 1, we will determine whether T cells that express a Treg T cell receptor differentiate along Treg cell pathway. Even though most T cells in Treg TCR Tg mice do not express Foxp3, they may be partially differentiated Treg cells. We will determine whether or not this is the case, and test the functionality of the Treg cells in a colitis mouse model and a spontaneous experimental autoimmune encephalomyelitis mouse model. In Aim 2, we will test the hypothesis that Foxp3+ cells are selected independently of each other in small niches. We will assess whether the addition of three Treg cell clones results in a corresponding increase in the number of Foxp3+ cells, and we will also determine whether we can augment the size of the selecting Treg niche by stimulating thymus epithelial cell growth. In Aim 3, we will identify the cell type from the thymus that expresses natural ligands to Foxp3+ T cells. We will isolate the different epithelial cells and dendritic cells from WT mice and determine which cell type has the ability to induce T cell receptor stimulation and Foxp3 expression. We will also identify natural ligands for regulatory T cells and develop mimotopes that stimulate them. PUBLIC HEALTH RELEVANCE: The absence of Foxp3+ regulatory T cells causes some of the most severe inflammatory diseases, with early childhood mortality that can only be treated by bone marrow transplantation. We have generated new strains of mice to study the development of Foxp3+ regulatory T cells. We expect that using these new tools we will learn about the requirements for the generation of Foxp3+ regulatory T cells.